CN113957022B - Preparation method and application of composite microbial agent - Google Patents
Preparation method and application of composite microbial agent Download PDFInfo
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- CN113957022B CN113957022B CN202111496954.2A CN202111496954A CN113957022B CN 113957022 B CN113957022 B CN 113957022B CN 202111496954 A CN202111496954 A CN 202111496954A CN 113957022 B CN113957022 B CN 113957022B
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 48
- 239000002131 composite material Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 46
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 41
- 239000002351 wastewater Substances 0.000 claims abstract description 38
- 229920002472 Starch Polymers 0.000 claims abstract description 33
- 239000008107 starch Substances 0.000 claims abstract description 33
- 235000019698 starch Nutrition 0.000 claims abstract description 33
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 40
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 35
- 244000063299 Bacillus subtilis Species 0.000 claims description 27
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 27
- 238000012258 culturing Methods 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 19
- 238000011218 seed culture Methods 0.000 claims description 14
- 241000195493 Cryptophyta Species 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
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- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 8
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- 230000001580 bacterial effect Effects 0.000 claims description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
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- 239000002244 precipitate Substances 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
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- 239000008272 agar Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 229960001031 glucose Drugs 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
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- 239000000843 powder Substances 0.000 claims description 3
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- 229910015667 MoO4 Inorganic materials 0.000 claims description 2
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- 238000012546 transfer Methods 0.000 claims description 2
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- 230000002195 synergetic effect Effects 0.000 abstract description 2
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
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- 235000002017 Zea mays subsp mays Nutrition 0.000 description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
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- 229910052757 nitrogen Inorganic materials 0.000 description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 6
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- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 6
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 4
- 239000003225 biodiesel Substances 0.000 description 4
- 235000013922 glutamic acid Nutrition 0.000 description 4
- 239000004220 glutamic acid Substances 0.000 description 4
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- 239000011573 trace mineral Substances 0.000 description 4
- 235000013619 trace mineral Nutrition 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 229960003237 betaine Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000012364 cultivation method Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 230000000243 photosynthetic effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 241000192542 Anabaena Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000194105 Paenibacillus polymyxa Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 229960004642 ferric ammonium citrate Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000004313 iron ammonium citrate Substances 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 235000014705 isoleucine Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000013335 mesoporous material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 235000008729 phenylalanine Nutrition 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
- C02F3/325—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae as symbiotic combination of algae and bacteria
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/341—Consortia of bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
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- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Hydrology & Water Resources (AREA)
- Biomedical Technology (AREA)
- Water Supply & Treatment (AREA)
- General Chemical & Material Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a composite microbial agent, which is prepared by mixing functional bacillus and microalgae, centrifuging and re-suspending; the invention also provides application of the composite microbial agent, which is used for removing organic matters in starch wastewater and preparing renewable energy sources. The preparation method of the invention utilizes the symbiotic synergistic effect between the functional bacillus and the microalgae to promote the mass proliferation of the microalgae and the functional bacillus, thereby effectively improving the biomass of the composite microbial agent, being applicable to the preparation of the composite microbial agent, and the prepared composite microbial agent is further applied to the removal of organic matters in starch wastewater and also can be used for the preparation of renewable energy sources.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a microbial agent, and in particular relates to a preparation method and application of a composite microbial agent.
Background
Biomass energy is known to be the most common renewable energy source on earth, which is a form of energy that stores solar energy in the form of chemical energy in the living body through photosynthesis of plants, and is called green energy. The microalgae have the characteristics of various types, high biological yield, rapid growth and propagation, short growth period, strong capability of self-synthesized grease, no occupation of agricultural land, strong environment adaptability and the like, and are considered to be ideal productivity organisms. And a great amount of water resources, carbon sources and nitrogen and phosphorus nutrient salts are needed in the growth process of the microalgae.
The starch waste water is waste water produced in the process of producing starch or starch sugar, glucose, starch derivative and other substances by using agricultural products such as corn, potato, wheat, rice and the like as raw materials, belongs to high-concentration organic waste water, contains a large amount of soluble starch, a small amount of protein and other substances, has high chemical oxygen demand and total nitrogen discharge, can cause environmental pollution problems such as water eutrophication, serious spoilage and deterioration if the waste water is directly discharged into environmental water, cannot effectively utilize organic matters in the waste water, and causes serious waste of resources, so that the starch waste water can be discharged after being treated. However, the starch wastewater treatment process has great difficulty and high cost, so people have conducted extensive researches on the treatment of pollutants such as ammonia nitrogen, phosphorus, total nitrogen and the like.
The growth of microalgae is connected with the treatment of starch wastewater, and microalgae cells can utilize substrates in the starch wastewater in the growth process to convert substances such as organic nitrogen and phosphorus in the wastewater into high-energy organic compounds required by the microalgae cells in three forms of horizontal phosphorylation, oxidative phosphorylation and photosynthetic phosphorylation, so that organic substances such as nitrogen and phosphorus in the starch wastewater are effectively consumed. The connection utilizes rich nutrient substances in the starch wastewater, promotes biomass productivity and can achieve the effect of double optimization.
When the traditional biotechnology and the physical and chemical technology remove organic matters in starch wastewater, on one hand, energy is consumed, and on the other hand, the activity and the advanced treatment of microorganisms are further maintained to complete the efficient removal of nitrogen and phosphorus elements. So far, a plurality of algae species, especially green algae species, capable of purifying water quality with high efficiency have been screened. However, algae grow relatively slowly and have little availability for certain more complex organics, organic nitrogen, in starch wastewater. Therefore, for starch wastewater containing a large amount of organic matters, additional measures are required to enhance the treatment efficiency.
The prior art, granted the Chinese patent of publication No. CN 110128181A, discloses a production device of algae bacteria fertilizer based on biogas slurry, can continuously produce the biogas slurry fertilizer containing probiotics and nutrients (N, P, K) by using the biogas slurry as a fermentation raw material, and can be used for culturing chlorella or photosynthetic bacteria, but does not specifically describe the removal effect of each nutrient substance of the biogas slurry wastewater, and does not describe the specific growth condition of algae bacteria in the wastewater and the research of mixed culture treatment of the algae bacteria; the Chinese patent of the issued publication No. CN 107629962A discloses a method for culturing microalgae by treating starch wastewater by using a honeycomb-like reaction chamber prepared by using a molecular imprinting mesoporous material, but no suggestion of co-culturing algae bacteria is given; the Chinese patent of the patent publication No. CN 104357327A discloses a method for culturing microalgae on a large scale by using bean product wastewater, which can save a large amount of water resources and nutrient salts, greatly reduce the culture cost of the microalgae, and effectively remove COD, TN, TP in the wastewater, but does not disclose a method for greatly improving the biomass of the microalgae.
Disclosure of Invention
The invention aims to provide a preparation method of a composite microbial agent, which can improve the growth speed of algae in starch wastewater when the prepared composite microbial agent is used, so that the effect of removing organic matters in the starch wastewater and simultaneously preparing renewable energy sources by the composite microbial agent is realized.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The preparation method of the composite microbial agent comprises the following steps of:
S1, sequentially carrying out slant culture, seed culture and fermentation culture on functional bacillus to a logarithmic phase, centrifuging to obtain a precipitate, and re-suspending to obtain functional bacillus mud; in addition, in the case of the optical fiber,
S2, culturing the microalgae in a high density manner to a logarithmic phase, centrifuging to obtain a precipitate, and re-suspending to obtain microalgae mud;
S3, taking functional bacillus bacterial mud and microalgae algae mud, mixing, centrifuging and re-suspending to obtain the compound microbial agent.
As one limitation, in step S1, the functional bacillus is bacillus subtilis, bacillus amyloliquefaciens, bacillus polymyxa or bacillus licheniformis;
The slant culture is carried out at 30-37 ℃ for 24-48h, the culture medium is LB solid culture medium, and the weight of agar in the culture medium is 2-5% of the total weight of the culture medium.
As another limitation, in the step S1, the seed culture is to transfer the functional bacillus strain obtained after the slant culture to a liquid seed culture medium for shake culture to obtain a first-stage seed liquid; and (3) inoculating the first-stage seed liquid into a new liquid seed culture medium for re-culture to obtain a second-stage seed liquid.
As a further limitation, the shake culture is carried out at a temperature of 30-37 ℃ for 24-36 hours at a rotating speed of 150-300rpm;
The culture medium is subjected to re-culture, the volume ratio of the inoculum size to the culture medium is 1:50, the temperature is 30-37 ℃, the time is 24-36h, and the rotating speed is 150-300rpm;
The liquid seed culture medium comprises the following components in parts by weight: 20-35 parts of anhydrous glucose, 8-15 parts of peptone, 3-8 parts of yeast extract powder, 3-6 parts of monopotassium phosphate, 2-3 parts of calcium carbonate and 2-3 parts of ammonium sulfate.
As a third limitation, in the step S1, the fermentation culture is performed at the temperature of 30-37 ℃ for 24-36 hours at the rotating speed of 150-300rpm;
The culture medium comprises the following components in parts by weight: 8-15 parts of peptone, 3-8 parts of yeast extract, 8-15 parts of sodium chloride and pH of 7.4;
the centrifugal speed is 4000-8000rpm, and the time is 3-10min;
the resuspension is three times in ultrapure water.
As a fourth limitation, in step S2, the microalgae are chlorella pyrenoidosa, anabaena, or Shan Qizao;
The high-density culture is carried out by culturing the sterile chlorella gamma at 23-30 ℃ with the illumination intensity of 2000-2800lux and the illumination period of 24 hours: culturing in BG11 liquid culture medium to logarithmic phase under the condition of 0h light-dark ratio to obtain sterile Chlorella psi;
the centrifugation is carried out at 6000-10000rpm for 8-15min;
the re-suspension is performed three times in ultrapure water;
The BG-11 culture medium comprises the following components in parts by weight: 100-200 parts of NaNO 3, 3-5 parts of K 2HPO4, 6-9 parts of MgSO 4·7H2 O, 3-4 parts of CaCl 2·2H2 O, 0.5-0.7 part of ammoniumcitrate, 0.5-0.7 part of ferr i cammonium citrate, 0.1-0.2 part of EDTA-Na 2, 1-3 parts of Na 2CO3 and 100-300 parts of A5 culture solution.
As a further limitation, the A5 culture solution includes, in parts by weight: 2-3 parts of H 3BO3, 1.5-2 parts of MnCl 2·4H2 O, 0.2-0.3 part of ZnSO 4·7H2 O, 0.3-0.5 part of Na 2MoO4·2H2 O, 0.05-0.1 part of CuSO 4·5H2 O and 0.03-0.09 part of Co (NO 3)2·6H2 O).
As a fifth limitation, in step S3, the volume ratio of the functional bacillus bacterial mud to the microalgae mud is 1:0.8-1.5;
the centrifugal speed is 4000-8000rpm, and the time is 3-10min;
The resuspension is performed three times in ultrapure water.
The invention also provides an application of the composite microbial agent, which is prepared by the preparation method of the composite microbial agent in any one of claims 1-8, and the composite microbial agent is used for removing organic matters in starch wastewater.
The invention also provides another application of the composite microbial agent, which is prepared by the preparation method of the composite microbial agent in any one of claims 1-8, wherein the composite microbial agent is used for preparing renewable energy sources-biodiesel.
The renewable energy biodiesel can be prepared by a method of methyl esterifying grease in chlorella by the compound microbial agent compounded by functional bacillus and microalgae, the principle of the utilization is that the microalgae converts light energy and CO 2 into grease through photosynthesis, and the grease and substances such as methanol are subjected to transesterification reaction to finally form biodiesel, so that the improvement of the total grease content in algae cells has important significance for the preparation of the microalgae biodiesel.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
① In the preparation method of the compound microbial agent, the selected functional bacillus can secrete a large amount of extracellular secretions such as glutamic acid, proline, tyrosine, glycine, citric acid, betaine and the like, and the extracellular secretions are microelements necessary for the growth of microalgae, so that the preparation method has the beneficial effects of promoting the growth and propagation of the microalgae;
② In the preparation method of the compound microbial agent, the selected microalgae can secrete a large amount of extracellular secretions such as phosphoric acid, inositol and the like, and the extracellular secretions are microelements necessary for the growth of functional bacillus, so that the preparation method has the beneficial effects of promoting the growth and propagation of the functional bacillus;
③ In the application of the composite microbial agent, microalgae and functional bacillus are mixed and cultured, and the biomass of the microbial agent is improved through cooperation and advantage complementation;
④ The composite microbial agent can fully utilize organic matters in the starch wastewater as nutrient substances required by self growth, and can be used for preparing renewable energy sources while effectively removing the organic matters in the starch wastewater.
The preparation method is suitable for preparing the composite microbial agent, and the prepared composite microbial agent is suitable for removing organic matters in starch wastewater and preparing renewable energy sources.
Drawings
The invention will be described in more detail below with reference to the attached drawings and specific examples;
FIG. 1 shows the results of extracellular secretion detection of the supernatant of Chlorella pyrenoidosa, the supernatant of Bacillus subtilis and the supernatant of the complex microbial agent ε in example 5 of the present invention;
FIG. 2 shows the results of the detection of the effect of extracellular secretion in the supernatant of Chlorella pyrenoidosa on the growth of Bacillus subtilis in example 5 of the present invention;
FIG. 3 shows the results of the detection of the effect of extracellular secretion in the supernatant of Bacillus subtilis in example 5 on the growth of Chlorella pyrenoidosa;
FIG. 4 shows the density of Bacillus and Chlorella in the mixed culture medium of functional Bacillus and Chlorella pyrenoidosa of example 6 of the present invention;
FIG. 5 shows the densities of Bacillus and Chlorella in the starch wastewater of functional Bacillus and Chlorella pyrenoidosa of example 6 of the present invention.
Detailed Description
The invention will now be described in further detail by way of specific examples, which are to be understood as illustrative only and not limiting.
Biological material:
Chlorella: from Alger Biotechnology Co., ltd;
Bacillus subtilis: from the laboratory of the fermentation technology innovation center (university of science and technology in Hebei province);
Example 1A method for culturing microalgae
The embodiment comprises the following steps sequentially carried out:
Taking sterilized chlorella gamma 10mL with the thallus density of 1X 10 6 cfu/mL, culturing in BG11 liquid culture medium at 26 ℃ under 2400lux illumination intensity for 24 hours: culturing for 0h to obtain sterile Chlorella psi culture solution after logarithmic growth phase, centrifuging the sterile Chlorella psi culture solution at 8000rpm for 10min, and re-suspending with ultrapure water for three times to obtain Chlorella pyrenoidosa mud, i.e. microalgae mud X 1;
wherein, the content of each component in the BG11 liquid medium is shown in table 1:
TABLE 1 BG11 liquid Medium formulation
| Main component | Concentration value |
| NaNO3(g/L) | 1.5 |
| K2HPO4(g/L) | 0.04 |
| MgSO4·7H2O(g/L) | 0.075 |
| CaCl2·2H2O(g/L) | 0.036 |
| Ammonium citrate(g/L) | 0.006 |
| Ferric ammonium citrate(g/L) | 0.006 |
| EDTA-Na2(g/L) | 0.001 |
| Na2CO3(g/L) | 0.02 |
| Microelement A5 solution (mL/L) | 1 |
The BG-11 solid culture medium alpha is prepared by adding agar accounting for 2% of the total weight of the BG11 liquid culture medium into the BG11 liquid culture medium, and uniformly mixing;
wherein the contents of the components in the trace element A5 solution are shown in Table 2.
TABLE 2 formulation of trace element A5 solution
EXAMPLES 2-6 cultivation method of microalgae
Examples 2-6 are a method for culturing Chlorella pyrenoidosa, which are basically the same as example 1, except that the raw materials and the process parameters are different, and the details are shown in Table 3:
Table 3 list of process parameters in examples 2-6
Wherein, the content of each component in BG11 liquid medium in examples 2-6 is shown in Table 4:
table 4 liquid culture Medium formulations of BG11 in examples 2-6
Wherein, the contents of each component in the trace element A5 solution in examples 2-6 are shown in Table 5:
Table 5 formulations of the trace element A5 solution in examples 2 to 6
The microalgae mud X 2-X6 is prepared in examples 2-6 respectively.
Example 7A method for culturing functional Bacillus
S1, strain slant culture: inoculating bacillus subtilis to LB solid slant culture medium containing 2% of agar by weight in streaking mode, and culturing at 37 ℃ for 24 hours to obtain strain delta;
S2, seed culture: transferring strain delta into a seed culture medium, shaking and culturing for 30 hours at 30 ℃ and 200rpm to obtain primary seed liquid, inoculating the primary seed liquid into a new seed culture medium according to 2% of inoculum size, and culturing for 24 hours at 37 ℃ and 200rpm to obtain secondary seed liquid;
S3, fermenting and culturing: inoculating the second-stage seed solution into LB culture medium according to the inoculum size of 2% volume ratio, fermenting and culturing at 35deg.C and 200rpm for 24h, centrifuging at 4000rpm for 10min after logarithmic growth phase, re-suspending with ultrapure water, and repeating for three times to obtain Bacillus subtilis bacterial mud, namely functional Bacillus subtilis bacterial mud Y 1;
Wherein, the LB culture medium comprises the following components: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, and the pH of LB medium is 7.4;
The seed culture medium comprises 30g/L of anhydrous glucose, 10g/L of peptone, 5g/L of yeast extract powder, 4.5g/L of monopotassium phosphate, 2.5g/L of calcium carbonate and 2.5g/L of ammonium sulfate.
Examples 8 to 12 cultivation method of functional Bacillus
Examples 8-12 are a method for culturing functional bacillus, which are basically the same as example 7, except that the raw materials and the process parameters are different, and the detailed details are shown in Table 6:
Table 6 list of process parameters in examples 8-12
Examples 8-12 respectively prepared functional bacillus bacterial sludge Y 2-Y6;
wherein the contents of each component in LB medium in examples 8 to 12 are shown in Table 7:
TABLE 7 LB medium formulations in examples 8-12
Wherein the contents of the components in the seed medium of examples 8 to 12 are shown in Table 8:
Table 8 seed Medium formulations in examples 8-12
Example 13 preparation method of Compound microbial agent
Taking 0.5L of functional bacillus bacterial sludge Y 1 with the density of 8 multiplied by 10 6 cfu/mL and 0.5L of microalgae alga sludge X 1 with the density of 1.1 multiplied by 10 5 cfu/mL, mixing, centrifuging for 10min at 6000 rotational speed, and taking the precipitate to be resuspended in ultrapure water for three times to obtain the composite microbial agent Z 1.
Preparation method of composite microbial agent of examples 14-18
Examples 14 to 18 are respectively a method for culturing a composite microbial agent, and the steps are basically the same as those of example 13, except that the raw material consumption and the process parameters are different, and the specific details are shown in Table 9:
table 9 list of process parameters in examples 14-18
The compound microbial agents Z 2-Z6 are prepared in examples 14-18 respectively.
Example 19 verification of treatment of corn steep liquor starch wastewater with composite microbial inoculant
S1, taking 3L of corn steep liquor starch wastewater, standing for 4 hours, removing precipitate, adjusting the pH value to 7, obtaining corn steep liquor starch wastewater A 1, and performing water quality test on the corn steep liquor starch wastewater A 1, wherein the test result is shown in Table 3:
Table 10 results of Water quality test of corn steep liquor starch wastewater A1
| Organic matter | COD(mg/L) | Total nitrogen (mg/L) | Total phosphorus (mg/L) | Ammonia nitrogen (mg/L) |
| Content of | 8098.83 | 531.86 | 164.15 | 600.824 |
300ML (10% of inoculum size) of the composite microbial inoculum Z 6 is inoculated into corn steep liquor starch wastewater A 1, the chlorella pyrenoidosa density in the corn steep liquor starch wastewater A 1 is controlled to be 1.1X10 5 cfu/mL, the bacillus subtilis density is 8×10 6 cfu/mL, the corn steep liquor starch wastewater A 1 is treated under the conditions of illumination intensity 2400lux, temperature 30 ℃ and rotating speed 140rpm, after 8 days, COD, TN, TP and ammonia nitrogen removal rates in the wastewater are measured by a quick spectrophotometry, a GB/T11894-89 alkaline potassium persulfate ultraviolet spectrophotometry, a GB/T11893-89 ammonium molybdate spectrophotometry and a Nahner reagent spectrophotometry, the COD removal rate is measured to be 90%, the total nitrogen removal rate is 82%, the total phosphorus removal rate is 78%, and the ammonia nitrogen removal rate is 64%.
EXAMPLE 20 verification of the synergistic action of functional Bacillus and Chlorella pyrenoidosa
Control group: chlorella pyrenoidosa (L.) Nees
Control two groups: bacillus subtilis
Experimental group: composite microbial agent Z 3
The experimental method comprises the following steps: a control group was placed in BG11 medium, cultured at 28℃and 140rpm under 2400Lux illumination intensity for 7 days, and centrifuged at 4℃and 10000rpm for 10min.
The control group and the experimental group are respectively placed in LB culture medium and BG11 culture medium, the control group is cultured for 24 hours at 37 ℃ and 200rpm, the experimental group is placed in BG11 culture medium, and the culture is carried out for 7 days at 28 ℃ and 140rpm under the condition of 2400Lux of illumination intensity. Respectively centrifuging culture solutions of the first control group, the second control group and the experimental group at 10000 rotation speed for 10min, collecting supernatant to obtain Chlorella pyrenoidosa supernatant, bacillus subtilis supernatant and compound microbial agent Z 3 supernatant, placing in liquid nitrogen, and storing for 20min, and detecting extracellular secretion in the three supernatants, wherein the detection result is shown in figure 1;
As can be seen from fig. 1, the extracellular secretion in the supernatant of chlorella pyrenoidosa is mainly phosphoric acid and inositol;
the extracellular secretion of the bacillus subtilis supernatant is mainly glutamic acid, proline, tyrosine, glycine, citric acid and betaine;
Extracellular secretion of the supernatant of the composite microbial agent epsilon mainly comprises glutamic acid, isoleucine, vitamin B6 and phenylalanine;
the extracellular secretion in the supernatant of the chlorella pyrenoidosa is respectively added into the bacillus subtilis culture solution by adopting a metabolism in vitro verification experiment, and the influence of the extracellular secretion in the supernatant of the chlorella pyrenoidosa on the growth of the bacillus subtilis is observed, and the result is shown in figure 2;
As is clear from FIG. 2, the growth of Bacillus was promoted at both the concentrations of 0.1g/L and 0.5g/L of phosphoric acid, whereas the promotion rate was 8% at the concentration of 0.1g/L and the improvement rate was 7% at the concentration of 0.5 g/L; the addition of inositol has a promoting effect on the growth of bacillus, when the concentration of the inositol is 1g/L, the growth speed of the bacillus is fastest, and when the concentration of the inositol is 8 hours after the addition of the inositol is 1g/L, the improvement rate of the growth of the bacillus reaches 30%;
The extracellular secretion in the supernatant of the bacillus subtilis is respectively added into the chlorella pyrenoidosa culture solution by adopting a metabolism in vitro verification experiment, the influence of the extracellular secretion in the supernatant of the bacillus subtilis on the growth of the chlorella pyrenoidosa is observed, and the result is shown in figure 3;
as can be seen from FIG. 3, when the concentration of added glutamic acid reaches 0.1g/L, the biomass of chlorella is improved by 94.3%; when the concentration of the added proline reaches 0.15g/L, the biomass of the chlorella is improved by 110%; when the concentration of the added tyrosine reaches 0.1g/L, the biomass of the chlorella is improved by 70.3 percent; when the concentration of the added glycine reaches 2g/L, the biomass of the chlorella is improved by 268 percent; when the concentration of the added citric acid reaches 0.1g/L, the biomass of the chlorella is improved by 16.7 percent; when the concentration of the added betaine is 0.05g/L, the biomass of the chlorella is improved by 15.08 percent.
EXAMPLE 21 efficacy verification of functional Bacillus and Chlorella pyrenoidosa Co-culture System
Blank one group: 100mL of BG11 medium of chlorella cultivated to logarithmic phase;
blank two groups: 100mL of bacillus subtilis LB medium cultured to logarithmic phase;
Experiment group: 100mL of chlorella cultivated to logarithmic phase and 100mL of bacillus subtilis BG11 cultivated to logarithmic phase;
experiment two groups: 100mL of chlorella cultivated to logarithmic phase and 100mL of starch wastewater solution of bacillus subtilis cultivated to logarithmic phase;
The experimental method comprises the following steps: placing the blank group I, the blank group II, the experiment group I and the experiment group II in a magnetic field illumination incubator with the illumination intensity of 2400Lux and 140rpm at the temperature of 28 ℃ for 5 days, taking out the incubator, and respectively measuring the density of microorganisms in the blank group I, the blank group II, the experiment group I and the experiment group II;
The density of chlorella in the blank group is 0.58 multiplied by 10 7cfu·mL-1;
In the blank two groups, the density of bacillus is 4.5×10 7cfu·mL-1;
The test results of one group of experiments are shown in FIG. 4, wherein the density of bacillus is 6.6X10 7cfu·mL-1, and the density of chlorella is 0.85X10 7cfu·mL-1; the density of bacillus is improved by 31.8% compared with that of bacillus in the blank two groups, and the biomass of the chlorella is improved by 46.6% compared with that of chlorella in the blank one group;
The test results of the second group are shown in FIG. 5, wherein the density of bacillus is 8.6X10 7cfu·mL-1, and the density of chlorella is 0.78X10 7cfu·mL-1; the density of bacillus is improved by 91.1% compared with that of the bacillus in the blank two groups, and the biomass of the chlorella is improved by 34% compared with that of the chlorella in the blank one group.
Example 22 Effect of Bacillus subtilis access time Point on Chlorella pyrenoidosa biomass
Inoculating chlorella cultivated to logarithmic phase into A, B, C, D, E culture mediums respectively, inoculating bacillus subtilis cultivated to logarithmic phase into five culture mediums respectively after 0h, 12h, 24h, 36h and 48h, wherein the inoculation amounts of chlorella and bacillus subtilis in the five culture mediums are the same, and detecting algal biomass in A, B, C, D, E five culture mediums after fermentation is finished to obtain optimal inoculation time, and the detection results are shown in table 4:
TABLE 4 Chlorella culture time Point access to Bacillus subtilis
| Culture medium | Access time (h) | Fermentation period (h) | Chlorella density (cfu/mL) | Bacillus subtilis Density (cfu/mL) |
| A | 0 | 168 | 3.5×107 | 1.42×107 |
| B | 12 | 336 | 2.96×107 | 1.02×107 |
| C | 24 | 192 | 4.2×107 | 2.15×107 |
| D | 36 | 120 | 3.23×107 | 1.25×107 |
| E | 48 | 100 | 2.75×107 | 0.8×107 |
As is clear from Table 4, the biomass of Chlorella obtained after inoculating Bacillus subtilis when the Chlorella was cultured for 24 hours was highest, wherein the density of Chlorella was 4.2X10 7cfu·mL-1 and the density of Bacillus subtilis was 2.15X10 7cfu·mL-1.
It should be noted that the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but the present invention is described in detail with reference to the foregoing embodiment, and it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (7)
1. The application of the composite microbial agent is characterized in that the composite microbial agent is used for removing organic matters in starch wastewater;
the preparation method of the composite microbial agent comprises the following steps in sequence:
S1, sequentially carrying out slant culture, seed culture and fermentation culture on functional bacillus to a logarithmic phase, centrifuging to obtain a precipitate, and re-suspending to obtain functional bacillus mud; in addition, in the case of the optical fiber,
S2, culturing the microalgae in a high density manner to a logarithmic phase, centrifuging to obtain a precipitate, and re-suspending to obtain microalgae mud;
s3, taking functional bacillus bacterial mud and microalgae algae mud, mixing, centrifuging and re-suspending to obtain the compound microbial agent;
the functional bacillus is bacillus subtilis;
The microalgae is Bifidobacterium parvifolium.
2. The use of a composite microbial agent according to claim 1, wherein in step S1, the slant culture is performed at a temperature of 30-37 ℃ for 24-48 hours, the culture medium is an LB solid culture medium, and the weight of agar in the culture medium is 2-5% of the total weight of the culture medium.
3. The application of the composite microbial agent according to claim 1, wherein in the step S1, the seed culture is to transfer the functional bacillus strain obtained after the slant culture is finished into a liquid seed culture medium for shake culture, so as to obtain a first-stage seed liquid; and (3) inoculating the first-stage seed liquid into a new liquid seed culture medium for re-culture to obtain a second-stage seed liquid.
4. The use of a composite microbial agent according to claim 3, wherein the shake culture is carried out at a temperature of 30-37 ℃ for 24-36 hours at a rotational speed of 150-300rpm;
The culture medium is subjected to re-culture, the volume ratio of the inoculum size to the culture medium is 1:50, the temperature is 30-37 ℃, the time is 24-36h, and the rotating speed is 150-300rpm;
The liquid seed culture medium comprises the following components in parts by weight: 20-35 parts of anhydrous glucose, 8-15 parts of peptone, 3-8 parts of yeast extract powder, 3-6 parts of monopotassium phosphate, 2-3 parts of calcium carbonate and 2-3 parts of ammonium sulfate.
5. The use of a composite microbial agent according to any one of claims 1-4, wherein in step S1:
the fermentation culture is carried out at the temperature of 30-37 ℃ for 24-36h and the rotating speed of 150-300rpm;
The culture medium comprises the following components in parts by weight: 8-15 parts of peptone, 3-8 parts of yeast extract, 8-15 parts of sodium chloride and pH of 7.4;
the centrifugal speed is 4000-8000rpm, and the time is 3-10min;
the resuspension is three times in ultrapure water.
6. The use of a composite microbial agent according to any one of claims 1-4, wherein in step S2:
The high-density culture is carried out by culturing the sterile chlorella gamma at 23-30 ℃ with the illumination intensity of 2000-2800lux and the illumination period of 24 hours: culturing in BG11 liquid culture medium to logarithmic phase under the condition of 0h light-dark ratio to obtain sterile Chlorella psi;
the centrifugation is carried out at 6000-10000rpm for 8-15min;
the re-suspension is performed three times in ultrapure water;
The BG-11 culture medium comprises the following components in parts by weight: 100-200 parts of NaNO 3, 3-5 parts of K 2HPO4 , 6-9 parts of MgSO 4·7H2 O, 3-4 parts of CaCl 2·2H2 O, 0.5-0.7 part of ammoniumcitrate, 0.5-0.7 part of ferri cammonium citrate, 0.1-0.2 part of EDTA-Na 2, 1-3 parts of Na 2CO3 and 100-300 parts of A5 culture solution;
The A5 culture solution comprises the following components in parts by weight: 2-3 parts of H 3BO3, 1.5-2 parts of MnCl 2·4H2 O, 0.2-0.3 part of ZnSO 4·7H2 O, 0.3-0.5 part of Na 2MoO4·2H2 O, 0.05-0.1 part of CuSO 4·5H2 O and 0.03-0.09 part of Co (NO 3)2·6H2 O).
7. The use of a complex microbial agent according to any one of claims 1-4, wherein in step S3, the centrifugation is performed at 4000-8000rpm for 3-10min;
The resuspension is performed three times in ultrapure water.
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